The present invention relates to prion proteins.
Prions are infectious pathogens that differ from bacteria, fungi, parasites, viroids, and viruses, both with respect to their structure and with respect to the diseases that they cause. Molecular biological and structural studies of prions promise to open new vistas into fundamental mechanisms of cellular regulation and homeostasis not previously appreciated. Kuru, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Strxc3xa4ussler-Scheinker syndrome (GSS) are all human neurodegenerative diseases that are caused by prions and are frequently transmissible to laboratory animals. Familial CJD and GSS are also genetic disorders. No effective therapy exists to prevent these fatal disorders2.
In addition to the prion diseases of humans, disorders of animals are included in the group of known prion diseases. Scrapie of sheep and goats is the most studied of the prion diseases. Bovine spongiform encephalopathy (BSE) is thought to result from abnormal feeding practices. BSE threatens the beef industry of Great Britain and possibly other countries; the production of pharmaceuticals involving cattle is also of concern. Control of sheep scrapie in many countries is a persistent and vexing problem2.
Since 1986, more than 170,000 cattle have developed BSE in Great Britain. Many investigators contend that BSE, often referred to as xe2x80x9cmad cow diseasexe2x80x9d, resulted from the feeding of dietary protein supplements derived from rendered sheep offal infected with scrapie to cattle, a practice banned since 1988. It is thought that BSE will disappear with the cessation of feeding rendered meat and bone meal, as has been the case in kuru of humans, confined to the Fore region of New Guinea and once the most common cause of death among women and children. Kuru has almost disappeared with the cessation of ritualistic cannibalism.
Prion diseases are associated with the accumulation of a conformational isomer (PrPSc) of host-derived prion protein (PrPc) with an increase in its xcex2-sheet content1. According to the protein-only hypothesis, PrPSc is the principal or sole component of transmissible prions2. Although the structure of PrPc has been determined3 and has been found to consist predominantly of xcex1-helices, the insolubility of PrPSc, which is isolated from tissue in a highly aggregated state and which has a high xcex2-sheet content, has precluded high-resolution structural analysis. Various workers have attempted to make forms of PrP which are intermediate between the normal (PrPc) form and the abnormal, pathogenic form (PrPSc), having a predominantly xcex2-sheet form therefore termed the xcex2-form.
Hornemann and Glockshuber PNAS 95, 6010-6014 (1998)8 describe a xcex2-intermediate which is an unfolding intermediate of mouse PrP and contains predominantly xcex2-sheet elements of secondary structure as opposed to xcex1-helix. Swietnicki et al (1997) J. Biol. Chem. 272:44, October 31 pp27517-27520 describe an identical folding intermediate derived from human PrP90-231. The mouse xcex2-intermediate is derived from oxidised PrP which contains the native disulphide bond. The mouse PrP intermediate required urea (a denaturant) for stabilisation. The reference on page 6011 xe2x80x9cResultsxe2x80x9d states that the mouse xcex2-intermediate exhibits stability at pH 4.0 in the absence of denaturant; however this is based upon an equilibrium calculation. The free energy of folding (Table 1, page 6012) is approximated from a fit of the equation described in Materials and Methods (page 6011) to the data in FIG. 1A. From this an equilibrium constant can be calculated which describes the small proportion of molecules that will exist as the xcex2-intermediate in the absence of denaturant. The proportion of molecules in this state is low (around 0.2%) and nothing can be said about their solubility in the absence of denaturant as they are not detectable. Indeed one would argue they are extremely unlikely to be soluble in the absence of denaturant because folding intermediates are structural states that are populated during rearrangement of a polypeptide chain from a random structure to a defined native conformation, or vice versa. They are characterised as having native-like secondary structure, few tertiary interactions, increased molecular volume, increased side chain mobility and exposed hydrophobic residues. These properties combined make them prone to aggregation and, as such, are generally insoluble in the absence of denaturants. Several references describe these properties in detail18-23.
Moreover, the above calculation is dependent upon the transition being a genuine equilibrium, ie. fully reversible. If the transition is not reversible this analysis is invalid. We have performed similar experiments and have found that full reversibility is abolished at protein concentrations in excess of 1 mg/ml, with refolding yields  less than 100%.
Zhang et al (1997) Biochem 36:12, 3543-3553 describe a xcex2-sheet form of recombinant Syrian hamster PrP containing residues 90-231 which is formed by a method involving refolding at a pH of 6.5. It is clear from page 3548, second column and FIG. 7, that the xcex2-form described is neither monomeric nor soluble in aqueous solution.
According to a first aspect the invention provides a method of making a xcex2-form of a prion protein which has more xcex2-sheet than xcex1-helix structure, can exist as a monomer and can retain solubility in aqueous solution in the s absence of a denaturant, the method comprising:
providing a reduced prion protein which does not include a disulphide bond and causing the conformation of the protein to change so that it adopts the xcex2-form.
Preferably, the change in conformation is caused by exposure to conditions of acidic pH, preferably a pH of 5.5 or less, more preferably a pH of 4.8 or less and most preferably a pH of 4.0.
Skilled persons will appreciate that the xcex2-sheet and xcex1-helix structure can be shown by circular dichroism spectropolarimetry as described herein. While the native prion protein state is characterised by a strong xcex1-helical signal, the xcex2-form of the invention shows a shift to a conformation dominated by xcex2-sheet. By xe2x80x9cdominatedxe2x80x9d in this context we include the meaning that there is more xcex2-sheet structure of the prion protein than xcex1-helix structure.
By xe2x80x9cexist as a monomerxe2x80x9d we include the meaning that the xcex2-form of the prion protein does not exist as an aggregate of two or more xcex2-form prion proteins. Skilled persons will appreciate that analytical sedimentation studies can be used to determine whether or not a protein exists in solution as a monomer or as an aggregate of two or more proteins. A suitable technique is described in Zhang et al (1997) Biochem, 36:12, 3542-3553 (see page 3545-3546 passage entitled Analytical Sedimentation). The technique involves the use of an analytical ultracentrifuge (Beckman Optimat XL-A) equipped with a six channel cell, using ultraviolet absorption between 220 and 280 nm.
By xe2x80x9ccan retain solubility in the absence of a denaturantxe2x80x9d we include the meaning that a significant proportion eg around 30% or more of the xcex2-form remains in solution as a monomer after centrifugation at 100,000 g for 1 hour and preferably 150,000 g for 8-16 hours, most preferably at 200,000 g for 8-16 hours. The centrifugation may be carried out on a 2 mg/ml aqueous solution of the xcex2-form prion protein comprising Na Acetate+10 mM Tris. HCl+pH 4.0 at 25xc2x0 C. The structural characteristics of the remaining protein in solution can be determined by circular dichroism spectropolarimetry, for example.
Preferably, the xcex2-form remains soluble without denaturant to a concentration of more than 1 mg/ml, more preferably at least 12 mg/ml, and especially more than 20 mg/ml.
It will of course be appreciated that the above requirement for the xcex2-form to be capable of retaining solubility in the absence of the denaturant in no way limits the invention to methods or compositions which do not include a denaturant.
A xcex2-form of a prion protein of the invention also comprises a prion protein which has at least 20% of its residues in xcex2-sheet structure, more preferably at least 50% and most preferably 50 to 60% or more, as determined by CD spectropolarimetry.
A xcex2-form of a prion protein of the invention also comprises a prion protein which is non-aggregated and exhibits partial resistance to proteinase K digestion.
A xcex2-form of a prion protein of the invention also comprises a prion protein which is non-aggregated but is capable of forming an aggregated fibrous and/or amyloid form, preferably on exposure to a denaturant.
Preferably, a xcex2-form of a prion protein of the invention also comprises a prion protein which is non-aggregated but is capable of forming a non-fibrillar aggregate on exposure to conditions of sufficient ionic strength. Preferably, the non-fibrillar aggregate is capable of forming a fibrillar structure.
By xe2x80x9cconditions of sufficient ionic strengthxe2x80x9d we mean an ionic strength capable of converting the non-aggregated xcex2-form to an aggregated form. For example, salt concentrations of 50 mM to 500 mM, especially 100 mM or more are sufficient to cause murine xcex2-form prion protein to form a non-fibrillar aggregate. A particularly preferred salt concentration is 100-200, more preferably 150 mM eg NaCl or KCl.
A xcex2-form of a prion protein of the invention also comprises a prion protein which is capable of interconverting between a xcex2-form as defined herein and an xcex1-form of a prion protein as described herein.
A xcex2-form of a prion protein of the invention may exhibit one or more of the above properties.
In another aspect, the invention provides a method of obtaining non-aggregated xcex2-form from a sample comprising partially digesting the sample with proteinase K.
It will be appreciated that by xe2x80x9cprion proteinxe2x80x9d is included variants, fragments and fusions that have interactions or activities which are substantially the same as those of a full length prion protein sequence, but which may be more convenient to use, for example in an assay. A xe2x80x9cvariantxe2x80x9d will have a region which has at least 70% (preferably 80, 90, 95 or 99%) sequence identity with the 91-231 region of native human PrP sequence described herein or the corresponding region in the PrP of other species as measured by the Bestfit Program of the Wisconsin-Sequence Analysis Package, version 8 for Unix. The percentage identity may be calculated by reference to a region of at least 50 amino acids (preferably at least 75, 100, 120 or 140) of the candidate variant molecule, and the most similar region of equivalent length in the native 91-231 region, allowing gaps of up to 5%.
The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Neddleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2.482. 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Bribskov and Burgess, Nucl. Acids Res. 14:6745, 1986 as described by Schwarts and Dayhoff, eds, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
Hybrid prion proteins comprising amino acid sequences from two or more different species also fall within the scope of the term xe2x80x9cprion proteinxe2x80x9d used herein. Hybrid proteins comprising protein domains from different species can be produced using known recombinant DNA techniques, such as those described in WO93/20093 in relation to hybrid human/porcine factor VIII proteins.
A xe2x80x9cfragmentxe2x80x9d comprises at least 50, preferably 75, 100, 120 or 130 amino acids of the native 91-231 sequence.
Such activities will include the abilities mentioned herein, such as the ability to be soluble without denaturant and may include the ability to raise antibodies and for use in screening compounds in accordance with the following aspects of the invention and the ability to form an aggregated fibrous and/or amyloid form especially a non-fibrillar aggregate which preferably comprises spherical particles having a diameter of from approx 10-20 nM which can be visualised by electron microscopy, when exposed to suitable conditions etc.
Preferably, the xcex2-form of a prion protein exhibits partial resistance to digestion with proteinase K(PK).
By xe2x80x9cpartial resistance to digestion with proteinase K (PK)xe2x80x9d we include the meaning that after incubation of 1 mg/ml of the protein in 10 mM NaAcetate+10 mM Tris. Acetate, pH 8.0 with 0.5 xcexcg/ml PK (based on the total digestion reaction volume) at 37xc2x0 C. for 30 mins some protein can be shown to be undigested when subjected to SDS-PAGE as described herein. Preferably, the majority of the protein is undigested.
Preferably, the xcex2-form of the invention displays resistance to digestion at increased concentrations of PK eg 5 xcexcg/ml PK or more.
The disease-related isoform of PrP, PrPSc, is distinguished biochemically from the normal cellular isoform of the protein, PrPc, by its partial resistence to digestion with the enzyme proteinase K. We have now demonstrated that not only aggregated xcex2-PrP is protease resistant but also that the soluble xcex2-PrP monomer is also PK-resistant and to a level approximating to that seen with PrPSc. This is strong evidence to support the contention that xcex2-PrP may be the precursor of PrPSc.
The novel xcex2-form, or an aggregate of two or more xcex2-forms, of the invention may be used to prepare antibodies which selectively recognise the xcex2-form (whether aggregated or not) rather than the xcex1-form or vice versa.
By xe2x80x9cxcex1-formxe2x80x9d of a prion protein we include the meaning of a prion protein which has more xcex1-helical than xcex2-sheet structure. The xcex1-form may also be characterised by sensitivity to degradation by proteinase K.
Any reductant and conditions which allow reduction can be used in the method of the invention as long as they do not cause irreversible modification to the polypeptide chain. Reduction of a disulphide bond can be determined by Ellman""s assay (Ellman, G. L., 1959, Arch Biochem and Biophys). Reduction of the disulphide bond preferably takes place before the pH is lowered. The acidic pH at which conformation change takes place may be approximately pH 5.5 or less, and preferably pH 4.8 or less, most preferably a pH of 4.0. Skilled persons will appreciate that any buffer that is effective around pH 4.0 can be used, such as 10 mM NaAcetate+10 mM Tris.Acetate.
Preferably, the xcex2-form has substantially the same molecular volume (measured by size exclusion chromatography) as the native form of the prion protein.
In a second aspect, the invention provides a preparation of a xcex2-form of a prion protein wherein at least 1% of the xcex2-form can exist as a monomer and can retain solubility in aqueous solution in the absence of a denaturant. Preferably, the xcex2-form is obtainable by a method according to the first aspect of the invention.
The invention also provides the above (soluble, undenatured) xcex2-form of a prion protein for use in medicine, preferably in the prevention, treatment and/or diagnosis of a prion disease.
It will be appreciated that by virtue of properties such as its solubility, the xcex2-form is amenable to high resolution structural analysis and so has particular utility for research into the mechanisms of prion disease especially prion replication. Such utility is not found in known insoluble forms of prion proteins.
The prion disease may be selected from one or more of the diseases affecting humans. Alternatively or additionally, the prion diseases are selected from one or more of the diseases which affect domestic farm animals such as cows, sheep and goats. Other prion diseases include transmissible mink encephalopathy; chronic wasting disease of mule deer and elk, bovine spongiform encephalopathy and, more recently, a whole series of new animal diseases that are thought to have arisen from their dietary exposure to the BSE agent. These include feline spongiform encephalopathy, affecting domestic cats and captive wild-cats (such as cheetahs, pumas, ocelots, tigers) and spongiform encephalopathies of captive exotic ungulates (including kudu, nyala, gemsbok, eland).
Preferably, the prion protein is selected from human, bovine or ovine prion proteins, more preferably human prion protein.
According to a third aspect of the invention there is provided a method of making an antibody against a prion protein having a xcex2-form as defined in accordance with the earlier aspects of the invention, comprising administering said xcex2-form to an animal and collecting and purifying the directly or indirectly resulting antibody. The antibody may be polyclonal, but is preferably monoclonal.
By xe2x80x9cantibodyxe2x80x9d in accordance with the invention we include molecules which comprise or consists of antigen binding fragments of an antibody including Fab, Fv, ScFv and dAb. We also include agents which incorporate such fragments as portions for targetting prion molecules and/or cells or viruses which display such molecules.
According to this aspect of the invention, there is also provided a monoclonal antibody capable of distinguishing between the native xcex1-form and the xcex2-form of a prion protein as defined in accordance with earlier aspects of the invention or vice versa. Also provided is a hybridoma cell capable of producing such a monoclonal antibody.
In accordance with this aspect of the invention there is also provided an antibody for use in medicine, which antibody binds preferentially to the xcex2-form of a prion protein rather than to the xcex1-form of the prion protein or vice versa. Preferably, the antibody is for use in the manufacture of a composition for use in the prevention, treatment and/or diagnosis of a prion disease.
According to a fourth aspect of the invention there is provided a method of detecting the presence of a prion protein having a xcex2-form as defined in accordance with the earlier aspects of the invention in a biological sample. The method preferably comprises providing an antibody preparation comprising an antibody which preferentially binds the xcex2-form rather than the xcex1-form and detecting whether the antibody binds xcex2-form.
Conveniently, the antibody is directly or indirectly labelled by suitable means and its binding to the xcex2-form is detected by detecting a label.
Preferably, the biological sample comprises or consists of a bodily fluid or tissue such as blood or blood derivative, ie a component such as plasma, lymphoid tissue (such as tonsils, appendices, lymph or spleen), cerebrospinal fluid faeces, urine, lymph or sputum. The biological sample may be a tissue sample eg a biopsy tissue sample.
It may be advantageous to introduce an anti-xcex2-form antibody into one of the tissues mentioned above either to detect xcex2-form or to remove xcex2-form before it reaches the brain. Such anti-xcex2-form antibodies are preferably antibodies which preferentially react with the xcex2-form rather than the normal xcex1-form of the prion protein.
By xe2x80x9cpreferentiallyxe2x80x9d according to the various aspects of the invention we include the meaning that the ratio of xcex1/xcex2 binding may be 45/55, 25/75, more preferably, 10/90, 5/95, 1/99 or substantially 0/100.
The invention also provides a method of detecting antibodies in a biological sample, which antibodies bind preferentially to a xcex2-form of a prion protein rather than the xcex1-form comprising exposing the xcex2-form to the biological sample to permit binding of antibody to the xcex2-form and detecting the binding of antibody to the xcex2-form. Optionally, the xcex2-form is immobilised before exposure to the sample.
The invention also provides a method of obtaining a xcex2-form binding agent which binds preferentially to a xcex2-form of a prion protein rather than an xcex1-form comprising exposing the xcex2-form to a sample to permit binding of agents to the xcex2-form and optionally collecting the agent bound to the xcex2-form. Optionally, the xcex2-form is immobilised before exposure to the sample. Preferably, the binding agent is directly or indirectly labelled and its binding to the xcex2-form is detected by detecting the label.
The invention also provides a kit useful for diagnosing a prion disease from a biological sample comprising a binding agent, preferably an antibody, which is capable of preferentially binding the xcex2-form rather than the xcex1-form, or a xcex2-form of a prion protein which binds said binding agent; and means for detecting binding of the binding agent to the xcex2-form. The binding agent or xcex2-form being coupled optionally to an inert support. Preferably, the means for detecting binding comprises a radioactive, enzymic or fluorescent label.
The invention also provides an in vitro method for diagnosing a predisposition to, or the presence of, a prion disease comprising providing a reduced xcex1-form of a prion protein, preferably at a pH of around 5.5 or less, preferably pH 4.8 or less, most preferably a pH of 4.0; comparing the amount or rate of formation of a xcex2-form as defined herein in the presence and absence of a biological sample eg from a patient. Increased rate or amount of xcex2-form formation is indicative of a predisposition to, or the presence of, a prion disease.
The invention also provides a method of treating a biological sample to remove a xcex2-form of a prion protein comprising providing a binding agent which binds preferentially to the xcex2-form of a prion protein rather than to the xcex1-form of the prion protein, exposing the biological sample to the binding agent whereby a xcex2-form of a prion protein can bind the binding agent and optionally collecting the treated biological sample. Preferably, the binding agent is immobilised before the exposure to the sample.
The invention also provides a method of diagnosing a predisposition to, or the presence of, a prion disease comprising providing a xcex2-form of a prion protein; providing a biological sample; and exposing the solution to the sample and detecting the presence of an aggregation of the xcex2-form, such an aggregation being indicative of predisposition to, or the presence of, a prion disease.
Preferably, the aggregation of the xcex2-form is a non-fibrillar aggregate which preferably comprises spherical or irregularly shaped particles having a diameter of from 10-20 nm which can be visualised by electron microscopy.
The invention also provides the use of a xcex2-form or a non-fibrillar aggregate thereof in the manufacture of a composition for use as a vaccine against a prion disease. A vaccine composition of the invention preferably comprises a xcex2-form or a non-fibrillar aggregate thereof and an adjuvant.
According to a fifth aspect of the invention there is provided a method of identifying an agent that is capable of preventing, reducing and/or reversing the conversion of a prion protein to a xcex2-form as defined above, the method comprising: providing a sample of a prion protein and comparing the amount of the xcex2-form quantitatively or qualitatively in the presence and absence of a test agent.
In a sixth aspect of the invention, there is provided a method of identifying an agent that is capable of preventing or reducing the conversion of a prion protein from the xcex2-form, as defined in accordance with earlier aspects of the invention, to an aggregated fibrous and/or amyloid form, especially a non-fibrillar aggregate mentioned above, the method comprising providing a solution containing the xcex2-form and comparing qualitatively or quantitatively the amount of the aggregated and/or amyloid form produced in the presence and absence of a test agent.
Preferably, the amount of the aggregated and/or amyloid, especially non-fibrillar aggregate, form is measured using a spectrofluorimeter.
In a seventh aspect of the invention there is provided an agent which is identifiable by a method as defined in accordance with the fifth or sixth aspect of the invention.
In an eighth aspect the invention provides an agent capable of preventing, reducing and/or reversing the conversion of a prion protein from an xcex1-form to a xcex2-form as defined in accordance with earlier aspects of the invention.
In a ninth aspect the invention provides an agent capable of preventing or reducing the conversion of a xcex2-form of a prion protein as defined in accordance with earlier aspects of the invention to an aggregated and/or amyloid , especially non-fibrillar aggregate, form.
The agents according to the seventh, eighth and ninth aspects of the invention may be a drug-like compound or lead compound for the development of a drug-like compound. Thus, the methods may be methods for identifying a drug-like compound or lead compound for the development of a drug-like compound that is capable of preventing, reducing and/or reversing the conversion of a prion protein to a xcex2-form; and/or that is capable of preventing and/or reducing the conversion of the xcex2-form to an aggregated and/or amyloid, especially non-fibrillar aggregate, form.
The term xe2x80x9cdrug-like compoundxe2x80x9d is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons molecular weight and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.
The term xe2x80x9clead compoundxe2x80x9d is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise, too toxic or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
The compounds identified in the methods of the invention may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds.
In another aspect the invention provides an agent that comprises a binding agent portion which binds preferentially to the xcex2-form of the prion protein rather than the xcex1-form, and an effector portion which is capable of one or more of the following functions: (1) preventing, reducing and/or reversing the conversion of a prion protein to a xcex2-form; (2) preventing or reducing the conversion of a prion protein from the xcex2-form to an aggregated fibrous and/or amyloid, especially a non-fibrillar aggregate form; or (3) destroying a xcex2-form of a prion protein and/or a cell or virus displaying such a protein.
Preferably, the binding agent portion comprise an antibody or a fragment thereof. Preferably the antibody or fragment thereof is made according to aspects of the present invention.
In one preferred embodiment the effector portion of an agent comprises a compound of the earlier aspects of the invention.
In another preferred embodiment the agent comprises an effector portion which is directly or indirectly cytotoxic.
By a xe2x80x9cdirectly cytotoxicxe2x80x9d portion we include a portion of an agent which is in itself toxic to the cell if it reaches, and preferably enters, the said cell.
By an xe2x80x9cindirectly cytotoxicxe2x80x9d portion we include a portion of an agent which can be converted into or produce a cytotoxic agent by the action of a further reagent, or which can convert a substantially non-toxic substance into a toxic substance. We also include a portion of an agent which can bind specifically to a compound which is directly or indirectly cytotoxic.
Non-limiting examples of cytotoxic portions include a drug, pro-drug, radionuclide, protein including an enzyme, antibody or any other therapeutically useful reagent, including cytokines such as tumour necrosis factor, interleukin-2 or interferon-xcex3.
Thus, the drug may be a cytotoxic chemical compound such as methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), daunorubicin or other intercalating agents. The protein may be ricin. The cytotoxic portion may comprise a highly radioactive atom, such iodine-131, rhenium-186, rhenium-188 or yttrium-90.
The enzyme, or enzymatic portion thereof, may be directly cytotoxic, such as DNaseI or RNase, or indirectly cytotoxic such as an enzyme which converts a substantially non-toxic pro-drug into a toxic form. The enzyme cytosine deaminase converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzyme thymidine kinase sensitises cells to treatment with the antiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276; Ezzedine et al (1991) New Biol 3, 608). The cytosine deaminase of any organism, for example E. coli or Saccharomyces cerevisiae, may be used. Examples of the construction of antibody-enzyme fusions are disclosed by Neuberger et al (1984) Nature 312, 604.
Other examples of pro-drug/enzyme combinations include those disclosed by Bagshawe et al (WO 88/07378), namely various alkylating agents and the Pseudomonas spp. CPG2 enzyme, and those disclosed by Epenetos and Rowlinson-Busza (WO 91/11201), namely cyanogenic pro-drugs (for example amygdalin) and plant-derived xcex1-glucosidases. The nitroreductase/CB1954 system described by Bridgewater et al (1995) Eur. J. Cancer 31A, 2362-2370 is another example of an enzyme/prodrug combination suitable for use in the invention.
In a tenth aspect the invention provides an agent in accordance with the earlier aspects of the invention for use in medicine. Preferably, use of the aspects in the manufacture of a composition for use in the prevention, treatment and/or diagnosis of a prion disease, or for use as a research reagent.
In an eleventh aspect the invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of an agent in accordance with the seventh, eighth and/or ninth aspects of the invention, together with a pharmaceutically acceptable diluent or carrier.
In a twelfth aspect the invention provides a method of preventing and/or treating a prion disease comprising administering to a subject an effective amount of an agent in accordance with the earlier aspects of the invention. By xe2x80x9ceffective amountxe2x80x9d we include the meaning that sufficient quantities of the agent are provided to produce a desired pharmaceutical effect beneficial to the health of the recipient.